{"paper_id":"2d2d0913-8300-4b0b-b2d2-7d1615724e20","body_text":"1 \n \nTitle \nMyonuclear domain-associated and central nucleation-dependent spatial restriction of dystrophin protein \nexpression in a novel DMD mouse model \n \nAuthors \nKatarzyna Chwalenia1,2, Vivi-Yun Feng1,2,3, Nicole Hemmer1,2,4, John C.W. Hildyard5, Liberty E. Roskrow5, \nRichard J. Piercy5, Eric T. Wang6,7, Annemieke Aartsma-Rus8, Maaike van Putten8, Matthew J.A. Wood1,4,9, \nThomas C. Roberts1,2,9,* \n \nAffiliations \n1 Department of Paediatrics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK \n2 Institute of Developmental and Regenerative Medicine, University of Oxford, IMS-Tetsuya Nakamura \nBuilding, Old Road Campus, Roosevelt Dr, Headington, Oxford OX3 7TY \n3 Freie Universität Berlin, Kaiserswerther Str. 16-18, 14195 Berlin, Germany \n4 Eberhard-Karls-Universität Tübingen, Geschwister-Scholl-Platz, 72074 Tübingen, Germany  \n5 Comparative Neuromuscular Diseases Laboratory, Department of Clinical Science and Services, Royal \nVeterinary College, Camden, London, NW1 0TU, UK \n6 Department of Molecular Genetics & Microbiology, Center for NeuroGenetics, Genetics Institute, University \nof Florida, Gainesville, FL, USA \n7 Myology Institute, University of Florida, Gainesville, FL, USA \n8 Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands \n9 MDUK Oxford Neuromuscular Centre, South Parks Road, Oxford, UK \n* To whom correspondence should be addressed. \n \nAuthor Contact Information \nKatarzyna Chwalenia  katarzyna.chwalenia@gmail.com \nVivi-Yun Feng   viviyun.feng@gmail.com \nNicole Hemmer   nic.hem@gmx.com \nJohn C.W. Hildyard  jhildyard@rvc.ac.uk \nLiberty E. Roskrow  lroskrow@rvc.ac.uk \nRichard J. Piercy   rpiercy@rvc.ac.uk \nEric T. Wang   eric.t.wang@ufl.edu \nAnnemieke Aartsma-Rus  A.M.Aartsma-Rus@lumc.nl \nMaaike van Putten  M.van_Putten@lumc.nl \nMatthew J.A. Wood  matthew.wood@idrm.ox.ac.uk \nThomas C. Roberts  thomas.roberts@idrm.ox.ac.uk  +44 1865 272159 \n \nORCID \nJohn C.W. Hildyard  0000-0003-2283-2118 \nLiberty E. Roskrow  0009-0006-3578-5962 \nRichard J. Piercy   0000-0002-4344-6438 \nAnnemieke Aartsma-Rus  0000-0003-1565-654X \nMaaike van Putten  0000-0002-0683-8897 \nMatthew J.A. Wood  0000-0002-5436-6011 \nThomas C. Roberts  0000-0002-3313-7631 \n \nCorresponding Author \nDr Thomas C. Roberts \nInstitute of Developmental and Regenerative Medicine  \nUniversity of Oxford \nIMS-Tetsuya Nakamura Building, \nOld Road Campus, Roosevelt Dr \nHeadington \nOxford \nOX3 7TY \nUnited Kingdom \nTelephone  +44 (0)1865 282833 \nEmail  thomas.roberts@idrm.ox.ac.uk\n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n2 \n \nAbstract \nThe restoration of uniformly-distributed dystrophin protein expression is an important \nconsideration for the development of advanced therapeutics for Duchenne muscular \ndystrophy (DMD). To explore this concept, we generated a novel genetic mouse model \n(mdx52-Xist\nΔ hs) that expresses variable, and non-uniformly distributed, dystrophin protein \nfrom birth as a consequence of skewed X-chromosome inactivation. mdx52-XistΔ hs myofibers \nare heterokaryons containing a mixture of myonuclei expressing either wild-type or mutant \ndystrophin alleles in a mutually exclusive manner, resulting in dystrophin protein being \nspatially restricted to corresponding dystrophin-expressing myonuclear domains. This \nphenotype models the situation in female DMD carriers, and dystrophic muscle in which \ndystrophin has been incompletely restored by partially-effective experimental therapeutics. \nTotal dystrophin expression increased in aged (60-week-old) mdx52-Xist\nΔ hs mice relative to \n6-week-old adults, suggestive of an accumulation of dystrophin-expressing myonuclei \nthrough positive selection, although this was insufficient to resolve sarcolemmal dystrophin \npatchiness. Nevertheless, compared to mice expressing no dystrophin, non-uniformly-\ndistributed dystrophin was protective against pathology-related muscle turnover in an \nexpression-level-dependent manner in both adult and aged mdx52-Xist\nΔ hs mice. Systematic \nclassification of isolated mdx52-XistΔ hs myofibers revealed profound differences associated \nwith central nucleation, with dystrophin found to be translationally repressed in centrally-\nnucleated myofibers and myofiber segments. These findings have important implications for \nthe development of dystrophin restoration therapies.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n3 \n \nIntroduction \nDMD is a monogenic muscle wasting disorder caused by pathogenic variants (frequently \nwhole exon deletions) in the DMD gene, which encodes the dystrophin protein. Dystrophin \nforms a mechanical link between the cytoskeleton (i.e. filamentous actin and microtubules) \nand the extracellular matrix via interactions with components of the dystrophin-associated \nprotein complex (DAPC), which consists of both structural and signalling proteins. Absence \nof dystrophin protein at the sarcolemma, and subsequent disruption of DAPC assembly, \nsensitizes muscle to contraction-induced damage.\n1 In DMD patients, this leads to perpetual \nmuscle turnover, persistent inflammation, and the progressive replacement of myocytes with \nfatty and fibrotic tissue. \n \nWhile still rare, the relatively high prevalence of the disorder (~1 in 5,000 males) and the \nseverity of the disease have made DMD a priority candidate for experimental therapeutics. \nIndeed, there are now four antisense oligonucleotide (ASO) drugs and one microdystrophin \ngene therapy that have received (accelerated) marketing authorization from the US FDA.\n2 \nFurthermore, a multitude of other approaches are under investigation, including CRISPR-\nCas9-mediated gene editing and upregulation of utrophin (a dystrophin paralogue).2–7 Despite \nthis progress, the clinical challenge of effectively treating DMD remains incompletely met, in \npart due to a combination of poor drug delivery, incomplete functionality of the restored \ninternally-deleted quasi-dystrophin protein, and failure to rescue dystrophin in all fibers. We \nhave observed that the pattern of dystrophin expression restored following treatment is \ndependent on the modality. Specifically, we observed a uniform sarcolemmal pattern of \ndystrophin expression following treatment of the mdx mouse model of DMD with peptide-\nphosphorodiamidate morpholino oligonucleotides (PPMOs) designed to induce exon skipping \nof Dmd exon 23 (containing a premature termination codon), 8,9 and a patchy pattern of \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n4 \n \ndystrophin expression following CRISPR-Cas9-mediated excision of the same exon in the \nseverely-affected dystrophin/utrophin double knock-out (dKO) mouse.10 Similar results were \nalso reported by Morin et al .11 Importantly, analysis of human biopsies has shown that \nincomplete sarcolemmal dystrophin coverage correlates with pathological severity in Becker \nmuscular dystrophy and intermediate muscular dystrophy patients (i.e. those with clinical \nphenotypes that are between those of BMD and DMD).\n12 Spatial restriction of dystrophin can \nbe attributed to its limited capacity to diffuse throughout the sarcolemma, such that it \nbecomes localized in the vicinity of its corresponding myonucleus of origin, consistent with \nthe myonuclear domain hypothesis.\n13 \n \nWe have previously modelled the effects of patchy sarcolemmal dystrophin expression using \nskewed X-chromosome inactivation (XCI) in a murine system. 9,14,15 To further investigate \nthis patchy dystrophin phenomenon, we have developed a novel mouse model that exhibits \npreferential XCI of the healthy X-chromosome, while the mutated X-chromosome carries a \npatient-relevant whole exon deletion of Dmd exon 52.\n16 Using this novel system, we show \nthat the female mdx52-XistΔ hs mice exhibit variable levels of dystrophin expression with a \ncharacteristic patchy pattern of dystrophin coverage at the sarcolemma. Comparison of \nmdx52-Xist\nΔ hs mice at adult (6 week) and aged (60 week) time points revealed an overall \nincrease in total dystrophin level, consistent with the accumulation of dystrophin-positive \nmyofibers with time. However, this increase in dystrophin expression was insufficient to \nresolve sarcolemmal dystrophin patchiness, suggesting that these fibers are incompletely \nprotected from the cycles of myonecrosis and compensatory regeneration that are \ncharacteristic pathological features of DMD. However, myofiber central nucleation was \ninversely correlated with total dystrophin expression, suggesting that patchy dystrophin \nexpression does offer myofibers a degree of protection. Interestingly, analysis of mdx52-\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n5 \n \nXistΔ hs isolated single myofibers revealed that centrally-nucleated myofibers and myofiber \nsegments were almost completely devoid of dystrophin or DAPC expression. This \nunexpected finding was attributed to local and specific inhibition of dystrophin expression at \nthe level of translation. This study has important implications for therapeutic efforts to restore \ndystrophin protein expression in Duchenne patients. \n \nResults \nDystrophin is expressed in a within-fiber patchy manner in adult mdx52-Xist\nΔ hs muscle. \nTo generate a genetic mouse model with patchy sarcolemmal dystrophin protein expression \nwe bred male mdx52 mice\n16,17 (which carry a patient-relevant deletion of Dmd  exon 52, \nleading to disruption of dystrophin expression) and female XistΔ hs mice 18 (which carry a \ndeletion in a DNase I hypersensitivity site within the Xist promoter, leading to skewed XCI of \nthe host chromosome). The resulting female F1 progeny ( mdx52-XistΔ hs) are expected to \nexpress dystrophin at variable levels as a consequence of skewed (i.e. preferential) XCI of the \nhealthy Dmd allele ( Figure S1).\n9,14,15 This mouse is a model of (i) female dystrophinopathy \n(previously known as manifesting carriers), 19,20 and (ii) the situation in dystrophic muscle \nfollowing partial CRISPR-Cas9 correction.9,10 \n \nAnalysis of mdx52-XistΔ hs females  (N=20) revealed a range of total dystrophin expression \nlevels in 6-week-old tibialis anterior (TA) muscles and mice were retrospectively assigned to \nhigh (~23-41% of WT dystrophin levels, n=4), medium (~11-17%, n=7), and low (~1-8%, \nn=9) dystrophin-expressing groups post-mortem, as determined by western blot ( Figure 1A-\nC). The distributions of dystrophin expression were consistent with those reported in similar \nstudies by our groups.9,14 Immunofluorescence analysis in the same tissues revealed a within-\nmyofiber patchy pattern of sarcolemmal dystrophin ( Figure 1D ). Regions of adjacent \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n6 \n \ndystrophin-positive and dystrophin-negative sarcolemma were observed in the mdx52-XistΔ hs \nmuscles at all dystrophin expression levels. By contrast, dystrophin was uniformly-distributed \nin age- and sex-matched wild-type C57 and Xist Δ hs mice, and absent in mdx52  controls \n(Figure 1D ). Patchiness was most apparent in longitudinal sections, but incomplete \nsarcolemmal coverage was also apparent in some myofibers in transverse sections (especially \nin the low dystrophin mdx52-XistΔ hs group). These data show that dystrophin mRNA and \nprotein are not free to diffuse freely within syncytial myofibers, consistent with previous \nreports.9–11 \n \nDystrophin, β -dystroglycan, and α -dystrobrevin are localized in sarcolemmal patches in \nmdx52-XistΔ hs isolated single myofibers. \nAnalysis of mdx52-Xist Δ hs isolated single extensor digitorum longus (EDL) myofibers \nrevealed similar patchy sarcolemmal distributions for dystrophin and the DAPC components \nβ -dystroglycan (DAG1) and α -dystrobrevin (DTNA) (Figure 2A). Dual staining showed that \ndystrophin and β -dystroglycan were co-localized to common regions of the sarcolemma, \nforming a ‘zebra-like’ banding pattern of staining ( Figure 2B ). Conversely, the DAPC \nprotein neuronal nitric oxide synthase (nNOS, NOS1) was uniformly distributed throughout \nsingle isolated myofibers derived from both mdx52-Xist\nΔ hs and WT C57 controls ( Figure \n2C).  \n \nDystrophin expression is inversely correlated with muscle histopathology in adult \nmdx52-Xist\nΔ hs muscles. \nHistopathological analysis in adult mdx52-XistΔ hs TA muscles revealed the presence of \nabundant centrally-nucleated fibers (CNFs) and foci of small diameter regenerating fibers \n(Figure 3A) at all levels of dystrophin expression, indicative of ongoing or historic muscle \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n7 \n \nturnover. Mean CNF values were 11.2%, 16.7%, and 38.7% for high, medium, and low \ndystrophin expressing muscles, respectively ( Figure 3B ). The percentage of CNFs was \nstrongly inversely correlated with dystrophin expression (Spearman’s r=-0.86, P=0.0023, \nFigure 3C). Myofiber size distributions were similar between all analysed genotypes (Figure \n3D). \n \nDystrophin patchiness is maintained in aged mdx52-Xist\nΔ hs muscle. \nIt has been proposed that dystrophin protein may accumulate with time following CRISPR-\nCas9-mediated correction as a result of positive selection of corrected, dystrophin-expressing \nmyofibers.4,5,10,21 To investigate this dystrophin accumulation phenomenon, we generated a \nseparate cohort of mdx52-Xist Δ hs female F1 mice ( N=21) and sacrificed them at 60 weeks of \nage. Dystrophin expression was determined in TA muscles by western blot in this ‘aged’ \ncohort and animals retrospectively assigned to high (53-91% of wild-type dystrophin, n=5), \nmedium (21-46%, n=8), and low (3-20%, n=8) dystrophin expression as described above \n(Figure 4A-C ). The mean dystrophin expression value for all aged mdx52-XistΔ hs animals \nwas ~2.8-fold higher than the mean of all 6-week-old mdx52-XistΔ hs animals ( P<0.001), \nconsistent with the enrichment of dystrophin positive myofibers as a consequence of positive \nselection. However, the patchy pattern of sarcolemmal dystrophin expression was maintained \nin aged animals at all dystrophin expression levels ( Figure 4C). Analysis of isolated single \nEDL myofibers from aged mdx52-Xist\nΔ hs showed that dystrophin, β -dystroglycan (DAG1), \nand α -dystrobrevin (DTNA) exhibited ‘zebra-like’ patchy immunostaining patterns, while \nthis effect was much less clear for nNOS (Figure 5), similar to those observations in adult \nanimals (Figure 2).  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n8 \n \nDystrophin expression is inversely correlated with muscle histopathology in aged \nmdx52-XistΔ hs muscles. \nHistopathological analysis in aged mdx52-XistΔ hs TA muscles revealed abundant CNFs and \nfoci of small diameter regenerating fibers ( Figure 6A) at all levels of dystrophin expression. \nMean CNF values were much larger than in the 6-weeks-old mdx52-XistΔ hs mice with 35.9%, \n51.1%, and 57.1% for high, medium, and low dystrophin expressing muscles, respectively \n(Figure 6B). The percentage of CNFs was inversely correlated with dystrophin expression \n(Spearman’s r=-0.5099, P=0.0257, Figure 6C ), although the correlation was substantially \nweaker than that observed for 6-week-old animals (Figure 3C ). Analysis of myofiber cross-\nsectional area revealed no differences between mdx52-Xist Δ hs groups ( Figure 6D). Together, \nthese results suggest that muscles expressing dystrophin in a patchy manner continue to \ndegenerate and regenerate throughout life, and that these pathological processes are to some \nextent ameliorated with higher levels of dystrophin expression. \n \nUtrophin expression does not correlate with patchy dystrophin levels in adult and aged \nmdx52-Xist\nΔ hs animals. \nUtrophin and dystrophin exhibit reciprocal expression patterns during muscle development \nand dystrophic pathology.\n22 Moreover, when expressed together, they bind to the same sites \nat the sarcolemma, as evidenced by electron microscopy detection of both proteins in muscles \nof transgenic mice.\n23 Due to the dystrophic nature of mdx52-XistΔ hs myofibers, and the \npresence of dystrophin in positive myonuclear domains, unrestricted availability of binding \nsites is expected within dystrophin-negative regions. We therefore reasoned that utrophin and \ndystrophin might exhibit reciprocal patterns of myonuclear domain restriction. \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n9 \n \nTo test this hypothesis, utrophin western blot was performed on muscle lysates from adult \nand aged mdx52-Xist Δ hs animals ( Figure S2A,B ). Utrophin expression was variable but \ndetected in all analysed animals, regardless of age or dystrophin levels. Expression of \nutrophin and dystrophin in TA muscles was not correlated for either 6-week-old or aged \nanimals (Spearman’s r = -0.2807, P = 0.6604, and r = -0.03506, P = 0.3023, respectively, \nFigure S2C,D).  \n \nTo assess the localization of utrophin in patchy dystrophin muscles, utrophin \nimmunofluorescence was performed in transverse TA sections from mdx52-Xist\nΔ hs animals \nidentified as exhibiting high utrophin expression within the low dystrophin expressing group \n(Figure S2E ). 12-week-old mdx52  tissue sections were used as control whereby a clear \nutrophin signal was detected at the membrane of relatively small, centrally-nucleated \nmyofibers organized in densely packed clusters ( Figure S2E). Accordingly, several small \ngroups of newly formed CNFs with positive utrophin staining were detected in aged mdx52-\nXistΔ hs muscle expressing low levels of dystrophin. Notably, no utrophin expression was \nobserved at the sarcolemma of larger mdx52-XistΔ hs TA myofibers ( Figure S2E). As such, \nutrophin expression in mdx52-XistΔ hs animals reflects the degree of ongoing muscle \nregeneration and is not concentrated in dystrophin-negative myonuclear domains. \n \nSerum microRNAs (miRNAs) have been investigated as minimally-invasive biomarkers in \nthe context of DMD. 24 In particular, we have previously reported that serum myomiR levels \nare inversely correlated with dystrophin expression levels following antisense \noligonucleotide-mediated exon skipping, suggesting that they may constitute promising \npharmacodynamic biomarkers.\n8,25 In 6-week-old animals, myomiRs were inversely correlated \nwith dystrophin expression level (Figure S3A-F). Conversely, in aged animals, there was no \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n10 \n \ndifference between serum myomiR levels between mdx52-XistΔ hs animals, and accordingly no \ncorrelation with dystrophin protein expression ( Figure S3G-L ). These observations are \nconsistent with our previous findings that serum myomiR levels are associated with \nregenerative pathology, which declines with age, and is effectively absent in aged \nanimals.\n26,27 \n \n \nDystrophin is not expressed in centrally-nucleated mdx52-XistΔ hs myofiber segments. \nInspection of single isolated myofibers revealed the existence of three types of fiber based on \nthe degree of central nucleation; (i) non-centrally-nucleated (59.9%), (ii) uniformly centrally-\nnucleated (16.6%), and (iii) segmented centrally-nucleated, whereby chains of centrally-\nlocated myonuclei were restricted to regions within the associated myofiber (23.5%) ( Figure \n7A). Non-centrally nucleated myofibers overwhelmingly (99.6%) exhibited patchy, ‘zebra-\nlike’ patterns of dystrophin distribution ( Figure 7B, and similar to micrographs in Figures 2 \nand 5A ). We next classified segmented fibers according to dystrophin/ β -dystroglycan (i.e. \nDAPC) expression, with signal in the non-centrally-nucleated region only (75.4%), coverage \nin both segments (18.4%), or no dystrophin/DAPC expression present at all (6.2%) ( Figure \n7C). Similarly, centrally-nucleated myofibers and myofiber segments were found to be \nalmost completely devoid of dystrophin/ β -dystroglycan expression. In fully centrally-\nnucleated myofibers, 88% contained no dystrophin or β -dystroglycan (i.e. DAPC) expression. \nThe remaining 12% of myofibers exhibited some expression, although this was frequently \nlimited to very small regions of membrane  (Figure 7D ). Importantly, in DAPC-positive \nfully-centrally-nucleated fibres, none exhibited the patchy, ‘zebra-like’ staining pattern. This \nfinding suggests that the observed dystrophin absence in centrally-nucleated regions is very \nunlikely to be driven by an XCI effect associated with the Xist\nΔ hs model.  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n11 \n \n \nThe absence of dystrophin and β -dystroglycan expression in centrally-nucleated myofiber \nsegments was even more apparent in segmented myofibers from aged (60-week-old) mdx52-\nXistΔ hs animals (Figure 8A), with aged fully-CNF myofibers being largely devoid of DAPC \nprotein expression (Figure 8B). A representative bulk preparation of myofibers from a single \n60-week-old mdx52-XistΔ hs illustrating this point is shown in Figure S4. Notably, the aged \nmyofibers were evidently hypertrophic and frequently contained multiple chains of centrally-\nlocated myonuclei. These observations indicate that centrally-nucleated myofibers in mdx52-\nXist\nΔ hs mice at this age are not recently regenerated. Immunostaining for the nuclear envelope \nmarker Lamin B1 (LAMB1) showed that these nuclei chains consist of intact myonuclei \nsquashed together, rather than fused together (Figure S5). \n \nTo assess whether muscle injury alone could induce a similar impairment in dystrophin \nexpression, we injected adult wild-type mice (20-30-week-old, both males and females) with \nthe muscle toxicant BaCl\n2 in order to induce acute myonecrosis and regeneration. Animals \nwere harvested after 29 days, after which the muscle morphology was restored but central \nnucleation persisted. Immunofluorescence staining in these animals revealed complete \nsarcolemmal dystrophin coverage, including in centrally-nucleated myofibers, suggesting that \nmuscle regeneration per se does not recapitulate the phenomenon of dystrophin absence in \ncentrally-nucleated myofiber segments observed in mdx52-Xist\nΔ hs mice (Figure S6). \n \nTaken together, these data show that dystrophin/the DAPC is largely absent in CNF \nmyofibers and in the centrally-nucleated regions of segmented myofibers. The absence of a \nsimilar effect in injured wild-type muscle suggests that this phenomenon is a feature of \ndystrophic muscle. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n12 \n \n \nTranslation of dystrophin is specifically impaired in centrally-nucleated myofiber \nsegments. \nThe absence of dystrophin in centrally-nucleated myofiber segments might be the result of a \nglobal impairment in either transcription or translation. RNA-FISH analysis using a pool of \nprobes spanning the Dmd transcript revealed puncta evenly distributed throughout mdx52-\nXist\nΔ hs single isolated segmented myofibers independent of dystrophin expression ( Figure \n9A). (Notably, this assay is not capable of distinguishing between the mutant and wild-type \nDmd alleles). Analysis of centrally-nucleated mdx52-XistΔ hs single isolated myofibers showed \nthat both Dmd  transcripts and titin (TTN) protein were uniformly distributed throughout all \nmyofibers assessed (Figure 9B ). Moreover, TTN exhibited a characteristic pattern of \nsarcomeric striation, indicative of myofiber maturity. TTN and filamentous Actin (F-Actin) \nwere found to be evenly distributed throughout both centrally-nucleated and non-centrally-\nnucleated myofiber regions, in stark contrast to the pattern observed for dystrophin ( Figure \n9C). These data demonstrate that there is no shortage or mislocalization of Dmd mRNAs in \ncentrally-nucleated myofiber regions, and that there is no local impairment in global protein \ntranslation. As such, these data suggest that dystrophin protein expression is specifically \nimpaired in mdx52-Xist\nΔ hs centrally-nucleated myofiber regions at the level of translation. \n \nMicrotubule network disruption is similar in dystrophin positive and negative segments. \nThe absence of dystrophin expression in centrally-nucleated regions might be a consequence \nof impaired mRNA trafficking following microtubule network disruption. One of the \nhallmarks of correct myofiber organization is the intricately organized microtubule network, \nwhich was recently shown to facilitate the active transport of various RNAs and proteins, \nincluding the ribosomal machinery, throughout the cell.\n28,29 Notably, dystrophin protein \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n13 \n \ncontains a microtubule-binding domain and therefore has been proposed to stabilize the \nmyofiber microtubule cytoskeleton.30,31 This role is supported by the fact that the microtubule \nnetwork is significantly disorganised in dystrophic mice, with costameric (transverse) \ncomponents being the most severely affected. 30,32 Thus, it is likely that non-uniformly \ndistributed dystrophin can modify the organization of the microtubules in mdx52-XistΔ hs \nmice, thereby partially facilitating correct mRNA transcript trafficking. TeDT (texture \ndetection technique) analysis of microtubules was performed on images acquired from \nmdx52-Xist\nΔ hs, mdx52, and wild-type C57 EDL myofibers ( n=40, 31, and 32 regions of \ninterest, respectively). The characteristic peak at the 90° intersection angle (representing the \ntransverse microtubules) together with a high vertical directionality score was detected in \nadult wild-type C57 myofibers ( Figure 10A-C ). In agreement with previous reports, the \nmicrotubule network was visibly disorganised in mdx52 animals, with a corresponding \nsignificant loss of transverse microtubules (Figure 10B,C).30,32 This disorganised pattern was \npartially restored in mdx52-XistΔ hs myofibers as represented by an intermediate distribution of \nmicrotubule intersection angles and vertical directionality scores ( Figure 10B,C ). These \nresults show that non-uniformly distributed dystrophin in the mdx52-XistΔ hs model is \nassociated with an intermediately distorted microtubule network. \n \nWe were next motivated to determine whether there was a difference between microtubule \nnetwork organization in dystrophin-positive and -negative mdx52-XistΔ hs myofiber segments \n(Figure 10D-F). No difference in microtubule lattice organization was observed between the \nanalysed domains in terms of microtubule intersection angle distribution ( Figure 10E ) or \nvertical directionality scores (Figure 10F). This suggests that the local absence of dystrophin \nalone may not be sufficient to induce cytoskeletal network disruption. \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n14 \n \nAged mdx52-XistΔ hs animals contain high proportions of hypertrophic centrally-\nnucleated myofibers in the absence of active regeneration. \nCentral nucleation is associated with muscle regeneration, but is known to persist long after \ninjury in mice (as long as 21 months).\n33,34 In addition, regenerating myofibers exhibit small \ncross-sectional areas and are positive for development-associated markers such as embryonic \nmyosin heavy chain and utrophin. 35 To better understand the phenomenon of dystrophin \nabsence in centrally nucleated myofibers/fiber segments, we analysed the extent of central \nnucleation in adult (6 week) and aged (60 week) mdx52-XistΔ hs TA muscle sections. \n \nCentral nucleation was shown to significantly increase in TA muscle sections from aged \nanimals (Figure 11A ). Furthermore, there was a shift towards a greater number of central \nmyonuclear chains with age in isolated EDL myofibers (Figure 11B ). Analysis of myofiber \ncross-sectional area revealed that aged mdx52-Xist\nΔ hs animals exhibited a pronounced shift \ntowards larger fibers ( Figure 11C), and that there was a statistically significant ( P<0.0001) \nshift in the mean Feret diameter in centrally-nucleated fibers ( Figure 11D). Taken together, \nthese data show that CNFs undergo substantial hypertrophy with age, which is likely driven \nby the progressive accretion of new myonuclei. Indeed, the number of nuclei per myofiber \nvolume was increased in CNF regions compared with non-CNF regions in mdx52 mice \n(Figure S7).\n36,37 These observations, together with the limited dispersion of central nuclei to \nthe myofiber periphery observed in mice,33,34 demonstrate that CNFs at later stages largely do \nnot constitute a population of recently formed muscle cells. 34,38 Together, these results \nemphasise the fact that the proportion of CNF at later stages of life in mice reflects the \ncumulative history of regeneration, and not recently regenerating immature muscle.\n38 The \nlatter point is important, because myofiber immaturity could be a potential explanation for the \nabsence of dystrophin in centrally-nucleated myofiber regions. Furthermore, the expression \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n15 \n \nof late-stage markers of muscle maturity (i.e. TTN, Figure 9B ) and the absence markers of \nearly-stage muscle development (i.e. UTRN, Figure S2) in CNFs lends further credence to \nthe notion that these myofibers are not recently regenerating and immature. \n \nNotably, in mature myofibers, nuclei play a crucial role as microtubule organization centers. \n39,40 As such, the distinct localization of myonuclei within CNFs and non-CNFs could \npotentially affect the organization of the microtubule network. To assess the contribution of \ncentral-nucleation to microtubule organization, cortical microtubules were analysed in CNF \nand non-CNF EDL myofibers harvested from 12-week-old mdx52 mice, as these muscles are \nexpected to contain very high proportions of centrally-nucleated myofibers.\n38 The \nmicrotubule network was visibly disorganized in CNFs ( Figure 11E ), which was \naccompanied by a quantitative decrease in transverse microtubules ( Figure 11F) and vertical \ndirectionality scores in CNFs ( P<0.0001) ( Figure 11G ). These results show, that within \ndystrophic myofibers, the microtubule lattice is substantially more disrupted in centrally-\nnucleated myofibers than in non-centrally-nucleated myofibers. \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n16 \n \nDiscussion \nThis study adds to the growing literature reporting spatial restriction of dystrophin protein to \nregions of sarcolemma proximal to its myonucleus of origin. Analyses in mdx52-XistΔ hs mice \nrevealed two distinct types of spatial phenomena. Firstly, a ‘zebra-like’ patchy pattern of \ndystrophin was observed in the majority of myofibers, as is expected from the underlying \npattern of skewed X-chromosome inactivation of the healthy Dmd  allele, and consistent with \nprevious observations ( Figures 1,2,4,5,8).\n9,11,14 An increase in overall dystrophin protein \nexpression levels was observed in aged vs. adult mdx52-XistΔ hs animals ( Figures 1C,4C ), \nconsistent with the notion that newly-dystrophin positive myofibers will tend to accumulate \nover time, as a consequence of a positive selection.\n4,41 Nevertheless, aged mdx52-Xist Δ hs \nanimals still exhibited non-uniform patterns of sarcolemmal dystrophin, indicating that \naccumulation of dystrophin-expressing regions is insufficient to completely resolve the \nobserved sarcolemmal patchiness ( Figure 5). These are disease-relevant observations which \nmirror situations in which dystrophic myofibers may exist as heterokaryons containing both \ndystrophin-expressing and non-dystrophin-expressing myonuclei. Specifically, in the case of \nfemale dystrophinopthay,\n41,42 and in dystrophic muscle after partially-effective dystrophin \nrestoration strategies. Our group recently showed that CRISPR-Cas9-mediated exon excision \nrestores dystrophin in a patchy manner along the sarcolemma, while PPMO-mediated exon \nskipping results in a uniform dystrophin distribution.\n8–10 The patchy pattern of dystrophin \nobserved for the former strategy was attributed to productive editing of the Dmd gene \noccurring in only a subset of myonuclei.10 As such, CRISPR-Cas9-treated dystrophic muscles \nare comprised of mosaic myofibers, a situation that is modelled by the mdx52-XistΔ hs mouse. \nSubsequently, Morin et al ., reported similar differential patterns of dystrophin restoration \nupon CRISPR-Cas9 gene editing or exon-skipping using tri-cyclo-DNA (tcDNA) \noligomers.\n11 Importantly, other types of dystrophin-restoration strategy have the potential to \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n17 \n \ngenerate myofiber heterokaryons, and consequently patchy sarcolemmal dystrophin coverage. \nFor example, in the case of cell therapy, dystrophin-expressing nuclei fuse with otherwise \ndystrophin negative myofibers.43–45 \n \nMyofibers expressing dystrophin in a patchy manner are likely to be susceptible to cycles of \ndamage and repair. This notion is supported by the observation that total dystrophin protein \nexpression and the proportion of centrally-nucleated fibers are inversely correlated in mdx52-\nXist\nΔ hs mice (Figures 3,6). This suggests that patchy dystrophin can, at least to some extent, \nprotect against the development of muscle histopathological features. These results are \nconsistent with previous reports in the mdx-XistΔhs and mdx/utrn−/−/XistΔ hs models, whereby \nanimals expressing low levels of patchy wild-type dystrophin showed higher proportions of \ncentrally-nucleated fibers in comparison to other groups.14,15 \n \nConcerning the second spatial phenomenon, we observed that dystrophin, and dystrophin-\nassociated proteins, were absent from centrally-nucleated myofibers and myofiber segments \n(Figures 7,8,9,S4 ). This surprising finding suggests that centrally-nucleated myofibers are \nrefractory to dystrophin expression, at least in this model. Our first thought was that \ndystrophin may be absent as a consequence of myofiber immaturity.46 However, several lines \nof evidence argue against this notion. Firstly, dystrophin absence in centrally-nucleated \nregions was observed in 60-week-old mice ( Figure 8,S4 ), when regeneration events are \nlikely to be very limited (as also evidenced by limited staining for utrophin, a marker of \nregenerating myofibers,47 Figure S2). Secondly, mdx52-XistΔ hs centrally-nucleated fibers also \nexhibit; (i) sizes consistent with healthy (or hypertrophic) myofibers ( Figures 10,11 ), (ii) \nexpression of late-stage markers of muscle differentiation like TTN ( Figures 8,9), and (iii) \nfrequently contained multiple chains of central nuclei ( Figures 8,9,11,S4,S5,S7). Together, \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n18 \n \nthese data suggest that these are in fact mature myofibers, exhibiting signs of repeated \nhistorical degeneration and accumulated repair. A second possible explanation is that the \nabsence of dystrophin in centrally-nucleated myofibers and myofiber segments is simply a \nconsequence of the XCI effect, whereby myonuclei that lack the capacity to expresses \ndystrophin have become clustered in the same region by chance. However, this explanation is \nnot supported by the data, as if this were true, we would expect to observe centrally-nucleated \nmyofibers that exhibit the ‘zebra-like’ patchy pattern of dystrophin throughout, of which we \nobserved none ( Figure 7,S4 ). As such, it is unlikely that central nucleation-associated \nabsence of dystrophin expression can be explained by model-associated XCI effects alone. \n \nMyoinjury by BaCl\n2 injection in wild-type mice did not recapitulate the effect observed in \nmdx52-XistΔ hs mice (i.e. post-regeneration, centrally-nucleated myofibers were uniformly \ndystrophin positive, Figure S6). As such, the absence of dystrophin in the centrally-nucleated \nmyofibers of mdx52-Xist Δ hs mice is likely the result of an interaction between the post-\nregeneration and dystrophic environments. In further support, the myofibers of X-linked \nmyotubular myopathy patients (and animal models thereof) exhibit centrally-nucleated \nmyofibers and express dystrophin.\n48 Similarly, centronuclear myopathy patients are also \nknown to express dystrophin, albeit with an abnormal intra-cytoplasmic localization. 49 These \nobservations suggest that central nucleation per se  is insufficient to prevent dystrophin \nexpression. \n \nDmd mRNA was found to be uniformly distributed throughout mdx52-XistΔhs myofibers (both \ncentrally-nucleated and non-centrally-nucleated regions) with all myonuclei containing \nnuclear ‘blobs’ that are characteristic of Dmd  RNA-FISH signal ( Figure 9A,B ). These \nfindings suggest that there is no impairment in Dmd  transcription in these regions. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n19 \n \nFurthermore, the uniform expression of TTN and F-actin proteins ( Figure 9B,C) suggests \nthat there is no global impairment in translation for centrally-nucleated regions. We therefore \nconclude that dystrophin is specifically repressed at the level of translation in mdx52-XistΔhs \ncentrally-nucleated myofibers/myofiber segments. Further work is needed to determine the \nmechanism of this repression, although it is tempting to speculate that local accumulation of \ntrans-acting factors, such as miRNAs, may be responsible. For example, miR-31 has been \nreported to repress dystrophin expression and is upregulated in DMD patient biopsies and \nmdx muscle tissues. 27,50,51 Likewise, other miRNAs (miR-146a, miR-146b, and miR-374a) \nhave also been reported to repress dystrophin.52,53 \n \nThe absence of dystrophin in centrally-nucleated myofibers/fiber-segments is a disease-\nrelevant observation, as it is suggestive of an additional challenge to the successful re-\nintroduction of dystrophin protein in dystrophic muscle, which may limit the effectiveness of \ncurrent and future experimental therapeutic interventions. Indeed, such a discrepancy \nbetween RNA-level exon skipping levels and dystrophin protein levels after antisense \noligonucleotide treatment in DMD patients has been previously reported. 54 Importantly, we \nhave previously reported widespread rescue of dystrophin expression after antisense \noligonucleotide-mediated exon skipping with highly potent PPMO compounds,8,9 and similar \nfindings have been reported by others using various dystrophin restoration strategies, 55–59 \nwhich would appear to contradict with the findings reported herein. Notably, treated animals \nare typically analysed using transverse muscle sections (with isolated single myofiber \nanalysis being relatively rare). As such, some within-fiber patchiness may have been \nobscured in these analyses. Alternatively, high levels of exon skipping may have been \nsufficient to overcome the mechanism that represses dystrophin protein expression in \ncentrally-nucleated fibers (i.e. there is a threshold effect). \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n20 \n \n \nThe microtubule network was found to be disrupted in mdx52-XistΔ hs mice at a level \nintermediate between that of C57 and mdx52 mice (Figure 10A-C). However, microtubule \nnetwork organization was found to be similar between dystrophin-positive and dystrophin-\nnegative myofiber regions in regions with ‘zebra-like’ patterns of dystrophin expression \n(Figure 10D,E ). Conversely, pronounced differences were observed in microtubule \norganization when comparing centrally-nucleated and non-centrally-nucleated fibers from \nmdx52 mice, suggesting that impaired trafficking of dystrophin mRNA and/or protein may \ncontribute to its translational repression in the case of the CNF-associated phenomenon \n(Figure 11E-G ). This aligns with previous findings by Percival et al. who demonstrated \naberrant distribution and increased density of Golgi elements at the surface of centrally \nnucleated wild-type fibres after cardiotoxin injury (in comparison to non-CNF).\n32 \n \nThe myonuclear domain theory posits that each nucleus within a syncytial myofiber controls \ngene expression and protein synthesis within a limited volume of surrounding sarcoplasm.\n13 \nThis concept is helpful for explaining some spatially-restricted gene expression features in \nsyncytial skeletal myofibers. Nevertheless, the definition of myonuclear domain is somewhat \nflexible (i.e. not restricted to a specific volume or sarcolemmal distance), but to a certain \nextent abstract and context dependent. Although the size of myonuclear domains can be \napproximated, it likely varies from cell to cell and protein to protein. Moreover, it is unclear \nhow the myonuclear domain theory would apply to centrally nucleated myofibers, which are \nnot only hypernucleated but also contain chains of seemingly compressed nuclei, that often \nrun in parallel to each other (Figure 8,S5, S7).  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n21 \n \nThe mdx52-XistΔhs model presents a unique opportunity to study dystrophin-dependent, and \nCNF-associated spatial phenomena in myofiber heterokaryons. However, it remains to be \ndetermined if such effects are present in DMD patient muscle. Dystrophin patchiness has \nbeen reported in female dystrophinopathy,\n41,42 and Torelli et al ., have reported an inverse \nrelationship between differential sarcoplasmic dystrophin coverage and disease severity in \npatient biopsies. 12 Notably, patient biopsy material is typically analysed in transverse \norientation, whereas dystrophin patchiness is more readily apparent in longitudinal sections. \nImportantly, centrally-located myonuclei are known to migrate to the myofiber periphery \nfollowing the completion of regeneration in human muscle, in contrast with the situation in \nmouse.33,34 However, experimentally determining whether central-nucleation-associated \nimpairment in dystrophin expression similarly occurs in human muscle may be challenging. \nAnalyses in healthy or DMD patient muscle would be inadequate, as these either express \n100% or close to 0% dystrophin (accounting for a small number of dystrophin-expressing \nrevertant fibers), respectively. The closest analogous situation would be that of female \ndystrophinopathy, or treated muscle with incomplete dystrophin restoration. Female \ndystrophinopathy is relatively rare (~2-22% of carrier females), 60,61 and there is a wide range \nof pathological presentation and XCI involvement,62 which would complicate these analyses. \nFurthermore, isolated single myofiber analyses in patient muscle are uncommon due to the \nrequirement for fresh material. Myofiber necrosis and regeneration are also known to be more \nprominent in mouse models than in DMD patients.63 \n \nIn conclusion, this work has identified two spatially-restricted dystrophin expression \nphenomena within the sarcolemma of a novel dystrophic mouse model. Local expression of \ndystrophin points to a previously unappreciated level of subcellular complexity in gene \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n22 \n \nexpression regulation with important implications for efforts to restore dystrophin protein \nexpression in the muscles of DMD patients. \n \nMethods \nAnimal studies \nAll experimental procedures were approved by the UK home office, under the project license \nnumber PP6777529 (Oxford) or PPL 70/7777 (RVC, approved by the Royal Veterinary \nCollege Animal Welfare and Ethical Review Board), in accordance with the Animals \n(Scientific Procedures) Act 1986. Animals were housed in individually ventilated cages with \na 12:12 hour light:dark cycle, with food and water provided ad libitum.  \n \nXist\n∆ hs animals were a kind gift from Prof. Neil Brockdorff (University of Oxford). 18 Xist∆ hs \nanimals contain a deletion of DNase hypersensitivity region upstream of the P1 promoter of \nthe Xist gene, resulting in preferential silencing of the mutation-containing chromosome. In \nheterozygous animals, the mutated X-chromosome is inactivated in up to 90% of the cells. 18 \nThe Xist∆ hs mouse has a mixed genetic background consisting of C57BL/6 and CBA. \n \nmdx52-Xist\n∆ hs animals were generated by crossing male mdx52 animals with female Xist∆ hs \nmice, with the resulting female F1 progeny used for experimentation. \n \nDystrophic mdx52 (C57BL/6J129S-Dmd\ntm1Mok) animals were a kind gift from Dr. Yoshitsugu \nAoki (National Centre of Neurology and Psychiatry, Tokyo, Japan). The line was generated \nby Dr. Motoya Katsuki via targeted replacement of exon 52 in the Dmd gene with a \nneomycin resistance transgene cassette (in the antisense orientation).16 \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n23 \n \nWild-type C57BL/6JOlaHsd (C57BL/6) mice were obtained from Inotiv (London, England) \nand served as wild-type control animals. Wild-type C57BL/10J mice were used for the BaCl2 \ninjury study. \nMyoinjury was induced by injection of 1.2% BaCl 2 (Sigma-Aldrich, MO, USA) in sterile \nsaline (total volume 20 µl) into TA muscles. Injections were performed under anaesthesia \nusing fentanyl/fluanisone (Hypnorm, Vetapharma, Leeds, UK) and midazolam (Hypnovel, \nRoche, Welwyn Garden City, UK), as described previously\n64. TA muscles were \nmacrodissected 29 days post injury, flash frozen in liquid nitrogen-cooled isopentane, and \nsamples stored at -80°C until ready for analysis. \n \nWestern blot \nDystrophin protein quantification was performed on tibialis anterior (TA) lysates. For protein \nextraction, 200 TA sections (8 µm thickness) were lysed in modified Radio-\nImmunoprecipitation Assay (RIPA) buffer (50 mM Tris pH 8, 150 mM NaCl, 1% IGEPAL \nCA-630, 0.5% sodium deoxycholate, 10% SDS) containing 1× cOmplete proteinase \ninhibitors (Merck, NJ, USA). Samples were heated for 3 min at 100°C and centrifuged at \nroom temperature for 10 minutes at 15,800 g. Protein concentration was measured using \nPierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) according to the \nmanufacturer’s instructions. \n \n20-40 µg of total protein were prepared in NuPAGE LDS sample buffer supplemented with \nNuPAGE sample reducing agent (both Thermo Fisher Scientific) and denatured for 10 \nminutes at 75°C. Standards were prepared as a mix of defined different protein ratios (0-75% \nof wild-type dystrophin protein levels) isolated from positive control, wild-type C57 and \nnegative control, dystrophic (mdx52) mouse TA. Linearity of signal was assumed for the few \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n24 \n \nsamples that fell outside of the standard range. All samples were loaded onto a pre-cast, \nNuPAGE Tris-Acetate (3-8%, Thermo Fisher Scientific) and electrophoresis run at 130 V for \n1 hour 45 minutes in NuPAGE Tris-Acetate SDS Running Buffer (Thermo Fisher Scientific). \nProtein was electrotransferred onto 0.45 µm polyvinylidene fluoride (PVDF) membranes \n(Merck) for 1 hour at 30 V followed by 1 hour at 100 V in 1× NuPAGE Transfer Buffer \n(Thermo Fisher Scientific) supplemented with 0.1 g/l of SDS (Sigma-Aldrich) and 20% \nmethanol. Total protein was visualized using a ChemiDoc Imaging system (Bio-Rad, CA, \nUSA) measuring fluorescence at 700 nm. The membrane was then washed in wash solution \nand blocked in blocking solution (either Odyssey blocking buffer (LI-COR Biosciences, NE, \nUSA) or 5% milk (w/v) in tris-buffered saline buffer supplemented with 0.15 Tween-20 (v/v, \nTBST)). Membranes were incubated with mouse anti-dystrophin, mouse anti-utrophin or \nmouse anti-vinculin primary antibody ( Table S1 ) overnight in blocking buffer at 4°C. \nMembranes were washed in tris-buffered saline buffer with 0.1% Tween-20 v/v (TBST) and \nincubated with anti-mouse IgG horseradish peroxidase (HRP) linked antibody ( Table S2) in \nblocking buffer + 0.1% Tween-20 for 1 hour at room temperature. Chemiluminescence signal \nwas detected using Clarity Western enhanced chemiluminescence (ECL) substrate (Bio-Rad). \nIf membrane re-probing was necessary for the detection of proteins of similar molecular mass \n(e.g. dystrophin and utrophin) or using different antibodies from the same host, the membrane \nwas stripped in 0.2 M sodium hydroxide (NaOH) for 30-120 minutes at room temperature. \nSubsequently, blocking step, primary and secondary antibody incubation and HRP-based \ndetection were performed as described above. \n \nSingle fiber isolation \nExtensor digitorum longus (EDL) single myofiber isolation was performed as described \npreviously.\n65 Briefly, EDL muscles were dissected tendon-to-tendon and incubated in 0.2% \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n25 \n \ncollagenase II (Worthington, NJ, USA) diluted in filter-sterilized DMEM (Thermo Fisher \nScientific, pre-warmed at 37°C) for 45-52 minutes at 37°C. Digestion was stopped by \ntransferring the muscle into a 3.5 cm cell culture dish, containing FluoroBrite DMEM media \n(Thermo Fisher Scientific) supplemented with 1% Antibiotic-Antimycotic (PSA: Penicillin, \nStreptomycin and Amphotericin B; Thermo Fisher Scientific) pre-warmed at 37°C. Single \nmyofibers were released from the muscle by gentle flushing using a 200 µl pipette under a \nstereomicroscope. Freshly isolated myofibers were transferred into a spot plate containing 4% \nultrapure paraformaldehyde solution (PFA, Electron Microscopy Sciences, PA, USA) for \nfixation for 10 minutes at room temperature. Fixed myofibers were washed twice with \nultrapure PBS for 5 minutes at room temperature. Immunofluorescence and/or hybridization \nchain reaction-based RNA in situ  hybridisation (HCR-RNA-FISH) were performed \nimmediately after fixation and PBS washes.  \n \nImmunofluorescence in tissue sections \nFresh frozen TA muscles were mounted onto corks with Tissue-TEK optimal cutting \ntemperature (OCT) Compound (Sakura, Japan) and cryosectioned (8 µm) in transverse and \nlongitudinal orientations. Samples were stored at -80°C prior to analysis. On the day of \nstaining, slides were air-dried and soaked in phosphate-buffered saline (PBS, Thermo Fisher \nScientific) for 10 minutes at room temperature. Sections were blocked in blocking buffer \ncomposed of PBS supplemented with 20% foetal calf serum (FCS, Thermo Fisher Scientific) \nand 20% normal goat serum (NGS, MP Biomedicals, CA, USA) for 2 hours at room \ntemperature. Subsequently, slides were incubated with primary antibodies (listed in Table \nS1) in blocking buffer for 2 hours at room temperature. After washing 3 times with PBS, \nslides were incubated with secondary fluorescent antibodies ( Table S2) in PBS or blocking \nbuffer for 1 hour at room temperature in darkness. Slides were then washed 3 times with \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n26 \n \nPBS, incubated with 4 /i3 ,6-diamidino-2-phenylindole (DAPI) or Hoechst in PBS (1:5,000, \nThermo Fisher Scientific), washed with PBS once more and mounted using Dako, \nFluorescence Mounting Medium (Agilent Technologies, CA, USA) or SlowFade Diamond \nAntifade Mountant (Thermo Fisher Scientific). \n \nImmunofluorescence in isolated fibers \nIf protein immunodetection and HCR RNA-FISH were performed on the same myofiber \nsample, staining for protein was carried out first. PFA-fixed myofibers were permeabilized \nwith 1% Triton-X100 (Sigma-Aldrich) for 10 minutes at room temperature followed by a \nsingle wash with ultrapure PBS (Thermo Fisher Scientific) for 5 minutes. Subsequently, \nblocking was performed for 30 minutes at room temperature with blocking buffer containing \neither 1% bovine serum albumin (BSA, Sigma-Aldrich) diluted in ddH\n2O if only protein \nimmunodetection was performed or 1% ultrapure BSA with RiboLock RNase Inhibitor at 1 \nU/µl (both Thermo Fisher Scientific) if protein detection was followed by RNA HCR-FISH \n(see below). Myofibers were then incubated with primary antibodies ( Table S1) diluted in \nblocking buffer for 2 hours at room temperature. Thereafter, myofibers were washed 3 times \nwith PBS (Thermo Fisher Scientific) containing 0.1% of Tween-20 (v/v, PBST, Sigma-\nAldrich) at room temperature. Subsequently, myofibers were incubated with secondary \nfluorescent antibodies (Table S2) for 2 hours at room temperature in the dark. \n \nIf only protein detection was performed, samples were washed 3 times with PBST and \nincubated with DAPI (Thermo Fisher Scientific) diluted in PBS for at least 2 minutes at room \ntemperature. Myofibers were transferred onto the SuperFrost Plus microslides (VWR) \ncontaining 35 µl of SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific) and \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n27 \n \ncovered with High Precision Cover Glasses, 1.5 mm thickness (Thorlabs, NJ, USA). Stained \nmyofibers were imaged on the following day and/or stored at -20 °C for repeated imaging. \n \nIf HCR-RNA-FISH was subsequently performed, samples were washed once with PBS and \nfixed in 4% PFA (Electron Microscopy Sciences) at room temperature. \n \nRNA-FISH \nRNA was detected using HCR RNA-FISH products purchased from Molecular Instruments \n(Los Angeles, CA, USA), following the generic sample in solution protocol with \nmodifications. PFA-fixed myofibers were washed twice with PBS (Thermo Fisher Scientific) \nfor 5 minutes at room temperature. Subsequently, samples were incubated with 2× ultrapure \nsaline-sodium citrate (SSC) buffer diluted in ultrapure H\n2O (both Thermo Fisher Scientific) \nfor 5 minutes at room temperature. Then, samples were incubated in pre-warmed (37°C) \nhybridization buffer (Molecular Instruments) for 30 minutes at 37°C. HCR probe sets were \nadded to fresh, pre-warmed (37°C) hybridization buffer at 1.25 nM/sample. Myofibers were \nincubated with the probe set solutions at 37°C in humidified conditions for 12-16 hours. \nSamples were washed with pre-warmed (37°C) wash buffer (Molecular Instruments) five \ntimes for 10 minutes at 37°C followed by two washes with 5\n/i3  SSCT (SSC supplemented \nwith 0.1% v/v Tween-20) at room temperature. Samples were subsequently incubated with \namplification buffer (Molecular Instruments) for 30 minutes at room temperature (pre-\namplification). Hairpin amplifiers were heated at 95°C for 90 seconds and cooled to room \ntemperature in the dark for 30 minutes. After pre-amplification, samples were incubated with \nhairpin amplifiers mixed in amplification buffer for 3.5-4 hour at room temperature in \ndarkness. Subsequently, samples were washed 5 times with 5× SSCT for 10 minutes and \nincubated with 0.1 µg/ml DAPI diluted in PBS (both Thermo Fisher Scientific) for at least 2 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n28 \n \nmin at room temperature. Myofibers were transferred onto the SuperFrost Plus microslides \n(VWR) and mounted using SlowFade Diamond Antifade Mountant (Thermo Fisher \nScientific) and High Precision Cover Glasses (Thorlabs). Stained myofibers were imaged on \nthe following day and/or stored at -20°C for repeated imaging. \n \nMicroscopy \nImmunofluorescence microscopy of tissue sections was performed using either wide-field \nLeica DMIRB Inverted Microscope with MetaMorph imaging software (Molecular Devices, \nCA, USA) or wide-field Leica DMi8 fluorescence microscope with LAS X Microscope \nScience Software Platform (all Leica Microsystems, Wetzlar, Germany). For each protein \nstaining, optimal exposure time was chosen based on negative staining control where samples \nwere incubated with secondary antibodies only, to account for background noise and \nautofluorescence of tissues. All images were processed using Fiji software.\n66 Standard image \nprocessing for tissue section images included background subtraction (based on rolling ball \nwith radius of 50 pixels), and brightness and contrast adjustment. \n \nSingle myofiber imaging was performed with ZEISS LSM 980 confocal microscope with \nAiryscan2 detector (ZEISS, Oberkochen, Germany). Depending on the application, the \nfollowing objectives were used: 40× Plan-Apochromat oil objective (numerical aperture NA \n= 1.4), 25× Plan-Apochromat (NA = 0.8) or 20× Plan-Apochromat (NA = 0.8). The choice of \nthe objective was based on the field of view and detail required in each experiment. \n \nImage Analysis \nCNF and CSA quantification in transverse tissue sections \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n29 \n \nImmunofluorescence was performed in fresh frozen TA sections using primary antibodies \nagainst α 2-laminin ( Table S1 ) to mark the muscle membrane, and DAPI (Thermo Fisher \nScientific) to label nuclei. The proportion of CNFs and myofiber cross-sectional area (CSA) \nwas analysed in transverse TA muscle sections using an open-source Fiji plugin: MuscleJ2, \naccording to developer’s instructions.\n67 2-5 whole TA sections were analysed per animal. For \nCNF proportion analysis, values for multiple sections from the same mouse were averaged. \n \nClassification of centrally nucleated, segmented and non-centrally nucleated myofibers \nClassification into non-centrally nucleated (non-CNF), segmented, and CNFs, and assessment \nof DAPC protein expression was performed on myofibers isolated from mdx52-Xist\n∆ hs mice \n(aged between 12 and 17 weeks). Myofibers were isolated and stained as described above, \nusing the antibodies listed in Table S1. Each slide, was scanned using the wide-field Leica \nDMi8 fluorescence microscope to visualize all myofibers. Each myofiber was visually \nexamined for nuclei (DAPI), and DAPC protein signal at the sarcolemma and classified \naccording to a pre-defined decision schema ( Figure S8). Briefly, myofibers were first \nclassified into non-CNF, segmented and CNF groups based on nuclear DAPI staining. Both \nmyofiber classes were further grouped into DAPC-expressing and non-expressing groups. \nDue to the observation that DAPC is present at the neuromuscular and myotendinous \njunctions of almost all mdx52-Xist\n∆ hs myofibers, junctional sarcolemma regions were \nexcluded from the analysis. \n \nAnalysis of the microtubule network organization \nMicrotubule intersection angle was analysed using TeDT direction v2017 according to the \ndeveloper’s instructions.68 Two to three cortical microtubule regions per myofiber segment \nwere analysed from z-stack images acquired at 40 × magnification. Pre-analysis image \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n30 \n \nprocessing included the z-projection of cortical microtubule region and background \nsubtraction (radius = 50 pixels). Images were arranged so that transverse microtubules were \npositioned at a 90° angle with respect to the longitudinal axis of the myofiber. The isotropic \nareas of microtubule nucleation surrounding myonuclei were excluded from the analysis. 40,68 \nIn total n = 60 regions from 32 myofibers, 79 regions from 40 myofibers, and 65 regions \nfrom 31 myofibers were analysed for C57, mdx52-XistΔ hs, and mdx52 mice respectively. The \nhistogram of proportion of microtubule directionality angles for each genotype was prepared \nusing averaged and 0-1 normalized values of fractions of microtubules at 0-176° in 4° \nintervals. \n \nSerum microRNA analysis \nSerum miRNA analysis was performed as described previously.\n69,70 Briefly, RNA was \nextracted from 50 µl blood serum samples using TRIzol LS (Thermo Fisher Scientific) \naccording to manufacturer’s instructions with minor modifications. A synthetic spike-in \ncontrol oligonucleotide with a non-mammalian miRNA sequence (i.e. cel-miR-39, 2.5 fmol, \nIDT) was added at the phenolic extraction phase in order to allow for between-sample \nnormalization. miRNAs were quantified using the small RNA TaqMan RT-qPCR method \nusing miRNA-specific stem loop reverse transcription primers. Details of miRNA assays are \nlisted in Table S3 . Reverse transcription was performed using the TaqMan MicroRNA \nReverse Transcription Kit (Thermo Fisher Scientific). cDNA was amplified using a StepOne \nPlus real-time PCR thermocycler with TaqMan Gene Expression Master Mix (both Thermo \nFisher Scientific) using universal cycling conditions: 95°C for 10 minutes, followed by 40 \ncycles of 95°C for 15 seconds and 60°C for 1 minute. All samples were analyzed in \nduplicate. Relative quantification was performed using the Pfaffl method,\n71 and miRNA-of-\ninterest abundance normalized to cel-miR-39.70 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n31 \n \n \nStatistical Analysis \nStatistical analyses were performed using GraphPad Prism (v10.2.3) (GraphPad Software \nInc., San Diego, California, USA). For comparisons of two groups, a Student’s t-test was \nused. For comparisons of more than two groups, an ordinary one-way analysis of variance \n(ANOVA) was performed with Bonferroni’s post hoc test for inter-group comparisons \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n32 \n \nAcknowledgements \nKC was supported by doctoral studentship from the Clarendon Fund in partnership with the \nMedical Research Council (MRC), and the Juel-Jenson Scholarship from St Cross College, \nOxford. This work was supported by a grant from the UK Medical Research Council \n(awarded to MJAW and TCR). \n \nAuthor Contributions \nTCR, KC, and MJAW conceived the study. TCR, RP, ETW, and MJAW supervised the \nwork. KC, VF, NH, JCWH, and LER performed experimentation. AAR and MvP provided \nessential reagents. TCR wrote the first draft of the manuscript. All authors contributed to the \nfinal version of the manuscript. \n \nDeclaration of Interests \nMJAW discloses being an advisor and shareholder in PepGen Ltd, a biotechnology company \nthat aims to generate exon skipping therapies for DMD. MJAW has filed multiple patents \nrelating to exon skipping technologies for treating DMD. AAR discloses being employed by \nLUMC which has patents on exon skipping technology, some of which has been licensed to \nBioMarin and subsequently sublicensed to Sarepta. As co-inventor of some of these patents \nAAR was entitled to a share of royalties. AAR further discloses being ad hoc consultant for \nPTC Therapeutics, Sarepta Therapeutics, Regenxbio, Dyne Therapeutics, Lilly, BioMarin \nPharmaceuticals Inc., Eisai, Entrada, Takeda, Splicesense, Galapagos, Sapreme, Italfarmaco \nand Astra Zeneca. AAR also reports being a member of the scientific advisory boards of \nHybridize Therapeutics (past), Silence Therapeutics, Sarepta therapeutics, Sapreme and \nMitorx. Remuneration for consulting and advising activities is paid to LUMC. In the past 5 \nyears, LUMC also received speaker honoraria from Alnylam Netherlands, Italfarmaco and \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n33 \n \nPfizer and funding for contract research from Sapreme, Eisai, BioMarin, Galapagos and \nSynaffix. Project funding is received from Sarepta Therapeutics and Entrada via unrestricted \ngrants. RJP has received funding for separate research programmes from Pfizer, Ultragenyx, \nand Exonics Therapeutics and has been a consultant to Exonics Therapeutics; the financial \ninterests were reviewed and approved by the University in accordance with conflict of \ninterest policies. The remaining authors declare no competing financial interests. \n \nKeywords \nDystrophin; myonuclear domain; central nucleation; DMD; X-chromosome inactivation \n \nData Availability Statement \nAll data are included in the manuscript. Raw data are available on request.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n34 \n \nReferences \n \n1. Petrof, B.J., Shrager, J.B., Stedman, H.H., Kelly, A.M., and Sweeney, H.L. (1993). \nDystrophin protects the sarcolemma from stresses developed during muscle contraction. \nProc. Natl. Acad. Sci. U.S.A. 90, 3710–3714. \n2. Roberts, T.C., Wood, M.J.A., and Davies, K.E. (2023). Therapeutic approaches for \nDuchenne muscular dystrophy. 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It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n41 \n \nFigure Legends \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n42 \n \nFigure 1 \nDystrophin expression is patchy in mdx52-Xist∆ hs muscle.  \n(A) mdx52-Xist∆ hs animals were assigned to high ( n=4), medium ( n=7), and low ( n=9) \ndystrophin expressing groups post-mortem based on protein quantification in 6-week-old \ntibialis anterior (TA) muscles as determined by (B) Western blot analysis. Wild-type C57and \nXist\nΔ hs mice were used as 100% dystrophin-expressing controls, and mdx52 mice used as \n~0% dystrophin-expressing controls. Vinculin was used as a loading control. ( C) Dystrophin \nwas quantified by comparison to standard curves containing defined mixtures of C57 and \nmdx52 TA lysates. ( D) Representative immunofluorescence staining of dystrophin and \nlaminin in transverse and longitudinal TA muscle sections of 6-week-old C57 wild-type, \nXist∆ hs, mdx52, and mdx52-Xist∆ hs animals from high, medium, and low dystrophin-\nexpressing groups. Within-fiber, patchy dystrophin expression resulting from skewed X-\nchromosome inactivation indicated with arrowheads. Scale bars indicate 100 µm, images \ntaken at 20× magnification. The percentage values indicate total dystrophin quantification in \nthe animals from which the sections were derived. Values are mean+SD. Statistical \nsignificance was assessed by one-way ANOVA with Bonferroni post hoc test, *** P<0.001, \n****P<0.0001.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n43 \n \n \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n44 \n \nFigure 2 \nDystrophin and DAPC protein expression is patchy in mdx52-XistΔ hs isolated single \nmyofibers.  \n(A) Representative immunofluorescence staining of β -dystroglycan ( β -DG), α -dystrobrevin \n(DTNA) and neuronal nitric oxide synthase (nNOS) in single isolated extensor digitorum \nlongus (EDL) myofibers of adult (6-12 weeks old) C57 (wild-type) and mdx52-XistΔ hs mice. \nRepresentative co-staining images of ( B) dystrophin and β -DG, and (C) β -DG and nNOS in \nthe isolated single myofiber of mdx52-Xist Δ hs animals. Scale bars indicates 20 µm, images \ntaken at 25× magnification. Nuclei were stained with DAPI. \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n45 \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n46 \n \nFigure 3 \nDystrophin expression is inversely correlated with histopathology in mdx52-XistΔ hs \nmuscle sections. \n(A) Representative immunofluorescence images of transverse TA muscle sections from adult \n(6-week-old) mdx52-XistΔ hs stained for laminin (green) as a sarcolemma marker and DAPI \n(blue) for nuclei visualisation. A magnified view of the sections shows the centrally nucleated \nfibers (CNF) proportion in two regions of the section. Tiled images were taken at 10× \nmagnification and stitched together using the LAS X software. Scale bars represent 100 µm. \n(B) CNF as a proportion of total myofibers analysed per TA section from mdx52-Xist\nΔ hs \nanimals expressing low, medium, and high dystrophin levels ( n=3). A single mdx52  animal \nwas included as a reference. ( C) Correlation analysis of dystrophin percentage and average \nCNF percentage in mdx52-XistΔ hs TA sections, with a single mdx52 animal as a reference. \nDystrophin protein percentage was derived from western blot analysis. ( D) Myofiber size \nvariability in TA sections of mdx52-Xist Δ hs animals ( n=3) visualized through proportion of \nfibers of specific cross-sectional area (CSA). Values are mean±SD. Statistical significance \nwas assessed by one-way ANOVA with Bonferroni post hoc  test relative to the Low \ndystrophin expressing group, **P<0.01. Nuclei were stained with DAPI.   \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n47 \n \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n48 \n \nFigure 4 \nDystrophin patchiness is maintained in aged mdx52-XistΔ hs muscle. \n(A) Aged mdx52-Xist ∆ hs animals were assigned to high ( n=5), medium (n=8), and low ( n=8) \ndystrophin expressing groups post-mortem based on protein quantification in 60-week-old \nTA muscles as determined by ( B) Western blot analysis. (C ) Dystrophin was quantified by \ncomparison to standard curves containing defined mixtures of C57 and mdx52 TA lysates. \nVinculin was utilised as a loading control. ( D) Representative immunofluorescence staining \nof dystrophin and laminin in transverse and longitudinal TA muscle sections of 60-week-old \nmdx52-Xist\n∆ hs animals from high, medium, and low dystrophin-expressing groups. Within-\nfiber, patchy dystrophin expression resulting from skewed X-chromosome inactivation \nindicated with arrowheads. Scale bars indicate 100 µm, images taken at 20× magnification. \nThe percentage values indicate total dystrophin quantification in the animals from which the \nsections were derived. Values are mean±SD. Statistical significance was assessed by one-way \nANOVA with Bonferroni post hoc test, ***P<0.001, ****P<0.0001.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n49 \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n50 \n \nFigure 5 \nDystrophin patchiness is maintained in aged mdx52-XistΔ hs isolated myofibers. \nRepresentative immunofluorescence co-staining of ( A) dystrophin and β -DG, (B) β -DG and \nDTNA, and ( C) β -DG and nNOS in isolated single 60-week-old mdx52-XistΔ hs EDL \nmyofibers. Tiled images were taken at 10× magnification and stitched together in LAS X \nsoftware. Scale bars indicate 100 µm. Nuclei were stained with DAPI. \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n51 \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n52 \n \nFigure 6 \nDystrophin expression is inversely correlated with histopathology in aged mdx52-XistΔ hs \nmuscle sections. \n(A) Representative immunofluorescence images of transverse TA muscle sections stained \nfrom aged (60-week-old) mdx52-XistΔ hs animals expressing high ( n=5), medium ( n=7), and \nlow ( n=7) dystrophin levels. Sections were stained for laminin (green) as a sarcolemma \nmarker and DAPI (blue) for nuclei visualisation. A magnified view of the sections shows the \ncentrally nucleated fibers (CNF) proportion in two regions of the section. Tiled images were \ntaken at 10× magnification and stitched together using the LAS X software. Scale bars \nrepresent 100 µm. ( B) CNF as a proportion of total myofibers analysed per TA section. ( C) \nCorrelation analysis of dystrophin percentage and average CNF percentage in analysed \nmdx52-XistΔ hs TA sections. ( D) Myofiber size variability in TA sections of mdx52-XistΔ hs \nanimals visualised through proportion of fibers of specific cross-sectional area (CNS). Values \nare mean±SD. Statistical significance was assessed by one-way ANOVA with Bonferroni \npost hoc test relative to the Low dystrophin expressing group, * P<0.05. Nuclei were stained \nwith DAPI. \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n53 \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n54 \n \nFigure 7 \nDystrophin is largely absent in centrally-nucleated myofibers/fiber segments. \n(A) Single isolated EDL myofibers harvested from adult (12-17 week old) mdx52-Xist\nΔ hs \nmice ( N=1,157) were sorted into non-centrally-nucleated (non-CNF), segmented (i.e. \ncontaining both centrally-nucleated and non-centrally-nucleated fiber regions), and fully \ncentrally-nucleated (CNF) categories. ( B) Non-CNFs (N=694) were further classified into \nthose with a ‘zebra-like’ patchy DAPC pattern of expression, and those with no DAPC \nexpression. ( C) Segmented myofibers ( N=272) were further classified based on whether \nDAPC proteins (i.e. dystrophin or β -dystroglycan) were detected in non-CNF segments, both \nCNF and non-CNF segments, or absent from both segments. ( D) Fully centrally-nucleated \nmyofibers (N=192) were classified based on whether or not DAPC proteins were detected. \nArrow heads indicate centrally-nucleated regions-of-interest. Asterisks indicate DAPC \npositive regions-of-interest. Tiled images were acquired at 10× magnification and stitched \nusing the LAS X software. Scale bars indicate 200 µm. Nuclei were stained with DAPI. \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n55 \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n56 \n \nFigure 8 \nDystrophin and DAPC protein expression is largely absent in centrally-nucleated \nmyofibers/fiber segments in aged mdx52-XistΔ hs mice. \nSingle EDL myofibers isolated from 60-week-old mdx52-XistΔ hs mice were analysed by \nimmunofluorescence and representative micrographs shown for (A ) dystrophin and β -\ndystroglycan ( β -DG) co-staining in segmented myofibers (i.e. containing both centrally-\nnucleated and non-centrally-nucleated regions), and ( B) dystrophin, α -dystrobrevin (DTNA) \nand β -DG in fully centrally-nucleated myofibers. Tiled images were acquired at 10× \nmagnification and stitched using the LAS X software. Scale bars represent 200 µm. Nuclei \nwere stained with DAPI.   \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n57 \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n58 \n \n \nFigure 9 \nDystrophin translation is suppressed in mdx52-XistΔ hs centrally-nucleated myofiber \nsegments. \nSingle EDL myofibers were isolated from adult (8-week-old) mdx52-XistΔ hs and analysed for \nimmunofluorescence and RNA fluorescence in situ hybridization (FISH). (A) Representative \nmicrograph of a single isolated EDL myofiber from an mdx52-XistΔ hs animal (12-week-old) \nshowing combined immunostaining for dystrophin protein and HCR-FISH for Dmd mRNA. \nImage taken at 40× magnification, scale bar represents 10 µm. (B) Representative micrograph \nof a centrally-nucleated myofiber stained for TTN protein and dystrophin mRNA. Tiled \nimages were acquired at 25× magnification and stitched together using the ZEN Blue \nsoftware. Scale bar represents 50 µm. ( C) Representative micrograph of a segmented \nmyofiber (i.e. containing both centrally-nucleated and non-centrally-nucleated regions) and a \nfully centrally-nucleated myofiber in the same frame stained for dystrophin, \nβ -dystroglycan \n(β -DG), and filamentous-actin (F-actin). Selected regions showing (i) a patchy, non-centrally \nnucleated segment, and (ii ) a centrally-nucleated segment, are enlarged and shown inset. \nImages were acquired at 25× magnification. Scale bars represent 20 µm. Nuclei were stained \nwith DAPI. \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n59 \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n60 \n \nFigure 10 \nmdx52-XistΔ hs myofibers exhibit intermediate microtubule network disorganization that \nis similar in dystrophin positive and negative segments. \n(A) Representative micrographs of immunostaining for α -tubulin to show cortical \nmicrotubule network organization in adult (8-12-weeks-old) C57 wild type, mdx52, and \nmdx52-XistΔ hs single isolated EDL myofibers. ( B) Histogram of mean distribution of \nmicrotubules of different intersection angles relative to myofiber long axis. The transverse, \ncostameric microtubule peak (90°) is marked with a dotted line. ( C) Vertical directionality \nscores reflecting the summed values of microtubules present between 80° to 100° within each \nmyofiber. (Sample sizes are; C57: n=60 ROIs, derived from 32 myofibers, mdx52: n=64 \nROIs, derived from 31 myofibers, mdx52-Xist\nΔ hs: n=79 ROIs, derived from 40 myofibers). \n(D) Representative micrographs of co-immunostaining for α -tubulin and dystrophin to show \ncortical microtubule network organization in a patchy dystrophin mdx52-XistΔ hs single \nisolated EDL myofiber. ( E) Histogram of mean distribution of microtubules of different \nintersection angles relative to myofiber long axis in dystrophin positive, and negative \nmyonuclear domains of mdx52-Xist\nΔ hs EDL myofibers. The transverse, costameric \nmicrotubule peak (90°) is marked with a dotted line. (Sample sizes are; DMD+: n =42 ROIs, \nderived from 24 myofibers, DMD-: n=40 ROIs, derived from 25 myofibers). ( F) Vertical \ndirectionality score reflecting the summed values of microtubules present between 80 to 100 \ndegrees within each fiber. Images taken at 40× magnification, scale bars represent 10 µm. \nPlotted values are mean±SD. Statistically significant differences were assessed by one-way \nANOVA with Bonferroni post hoc  test or two-tailed Student’s t-test, as appropriate. \n**P<0.01, **** P<0.0001. Nuclei were stained with DAPI. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n61 \n \n \n \n  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint \n\n62 \n \nFigure 11 \nCentral nucleation accumulates with age in mdx52-XistΔ hs mice and is associated with \nmicrotubule network disruption. \nAdult (6-week-old) and aged (60-week-old) mdx52-XistΔ hs TA muscle sections were \ncompared for (A) the percentage of CNF myofibers, ( B) the number of chains per centrally-\nnucleated myofiber, ( C) mean Feret diameter of CNFs, and ( D) Distribution of CNF cross-\nsectional area (CSA). Separately, adult (12-week-old) mdx52 single isolated EDL myofibers \nwere harvested and analysed for microtubule network organization in centrally-nucleated and \nnon-centrally-nucleated myofibers. (E) Representative micrographs of immunostaining for α -\ntubulin. (F) Histogram of mean distribution of microtubules of different intersection angles \nrelative to myofiber long axis in centrally-nucleated and non-centrally-nucleated mdx52 \nmyofibers. The transverse, costameric microtubule peak (90°) is marked with a dotted line. \n(G) Vertical directionality score reflecting the summed values of microtubules present \nbetween 80 to 100 degrees within each fiber. (Sample sizes are; CNF: n=44 ROIs, derived \nfrom 22 myofibers, non-CNF: n =60 ROIs, derived from 27 myofibers). Images taken at 40× \nmagnification, scale bar represents 10 µm. Values are mean±SD. Statistically significant \ndifferences were assessed by Student’s t -test, **P<0.01, ***P<0.001, ****P<0.0001. Nuclei \nwere stained with DAPI. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 24, 2025. ; https://doi.org/10.1101/2025.07.24.666562doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}